Equilibrium centrifugation of proteins in acid solutions. Ovalbumin in aqueous acetic, propionic, and butyric acids.

The use of short-chain aliphatic acids in conjunction with the partial specific volume of the native protein, v?_p for molecular weight estimations and for the study of macromolecular interactions was further tested: Equilibrium centrifugation of ovalbumin has been carried out in aqueous acetic, propionic, and butyric acids in the absence of any other added electrolyte. Under these experimental conditions there are two potential sources of error associated with the use of v?_p. These are: (a) preferential interaction with solvent components[$\text{Γ = (}\text{δm}{}_3\text{δm}{}_2\text{)μ}{}_1\text{,μ}{}_3\text{T, P}$] and (b) volume changes accompanying the exposure of the protein to acid (δv?_p). Volume changes associated with the transfer of ovalbumin to acid solutions have been measured. With this information at hand, it was possible to show that δv?_p and Γ in these solvent systems are both small; therefore, v?_p can be used instead. This work has also shown that the behavior of ovalbumin is distinct from that of β-lactoglobulin and aldolase. Thus, it appears that each protein has a characteristic behavior in these aqueous organic solvents which is related to some aspect of protein structure. Ovalbumin undergoes a series of reversible and irreversible interactions in these solvent systems which are dependent on both the length of the acid's carbon chain and the concentration of acid. An effort has been made to elucidate these interactions in terms of simple models.     

Some dilute-solution parameters of the levan of streptococcus salivarius in various solvents

The intrinsic viscosities, weight-average molecular weights (M?_w), and radii of gyration [($\text{R}{}^2{}_{\mathrm{g}}$)^{$\text{1}\text{2}$}≈] of Streptococcus salivarius levan in various solvents were respectively obtained from viscosity and light-scattering measurements. The data showed that the levan in water is not aggregated by hydrogen bonds, and that the values of both the refractive index and ($\text{R}{}^2{}_{\mathrm{g}}$)^{$\text{1}\text{2}$} are lower in water than in aqueous solutions of urea. Urea may break intramolecular hydrogen-bonds, e.g., between branches, allowing the molecule to expand.     

Preferential solvation of methylmercurated calf thymus deoxyribonucleic acid.

The changes in polymer-solvent interactions that occur when native calf thymus DNA is dialyzed against Na₂SO₄ solutions of a given ionic strength and buffer concentration but of varying concentrations in methylmercuric hydroxide have been investigated with the help of solution density measurements at 25 °C and pH 6.8–7.0. From measurements executed under equilibrium dialysis conditions at the three salt levels 5 mm, 0.05 m, and 0.5 m Na₂SO₄ (m refers to molality) and in the presence of 5 mm cacodylic acid buffer, the density increments $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ for native calf thymus DNA were determined as a function of CH₃HgOH concentration. $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ was found not to vary with organomercurial concentration, irrespective of the concentration of supporting electrolyte, until a certain CH₃HgOH concentration level has been reached, viz., , beyond which $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ increases strongly with increasing concentration of CH₃HgOH. As is shown by optical melting, $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ becomes a function of organomercurial concentration the moment DNA undergoes denaturation brought about by the complexing of CH₃HgOH with the various N-binding sites of the base residues in the DNA double helix.Polymer-solvent interactions, expressed in terms of preferential water interactions (“net hydration”) and preferential salt interactions (“salt solvation”), were derived from the $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ data in combination with data obtained on the preferential interaction of CH₃HgOH with denatured DNA and data on the partial specific volumes of all major solution components, gathered from density measurements on solutions with fixed concentrations of diffusible components. Evidence is presented which shows that denaturation in general decreases the net hydration while salt becomes preferentially associated with the polyelectrolyte. This process is further amplified by the interaction of CH₃HgOH with denatured DNA: Methylmercurated DNA alters the redistribution of diffusible components at dialysis equilibrium to such an extent that in a formal sense large amounts of water are rejected from the immediate vicinity of the polymer. The molecular implications of these findings are explored. The results are further discussed in the light of previous findings where the methylmercury-induced denaturation of DNA had been studied with the help of buoyant density measurements in a Cs₂SO₄ density gradient and by velocity-sedimentation in a variety of sulfate media.     

Vacuum ultraviolet circular dichroism spectrum of beta-turn in solution.

The vacuum ultraviolet circular dichroism spectrum of an isolated 4 → 1 hydrogen bonded β-turn is reported. The observed spectrum of N-acetyl-Pro-Gly-Leu-OH at ? 40°C in trifluoroethanol is in good agreement with the theoretically calculated CD spectrum of the β-turn conformation. This spectrum, particularly the presence of a strong negative band around 180 nm and a large ratio $\text{[θ]}{}_{201}\text{[θ]}{}_{225}$, can be taken as a characteristic feature of the isolated β-turn conformation. These CD spectral features can thus be used to distinguish the β-turn conformation from the β-structure in solution.     

The influence of charge on phosphatidic acid bilayer membranes

A complete titration of phosphatidic acid bilayer membranes was possible for the first time by the introduction of a new anaologue, $\text{1,2-}\text{dihexadecyl}\text{-sn-}\text{glycerol-3-phosphoric}$ acid, which has the advantage of a high chemical stability at extreme pH values. The synthesis of this phosphatidic acid is described and the phase transition behaviour in aqueous dispersions is compared with that of three ester phosphatidic acids; $\text{1,2-}\text{dimyristoyl}\text{-sn-}\text{glycerol-3-phosphoric}$ acid, 1,3-dimyristoylglycerol-2-phosphoric acid and $\text{1,2-}\text{dipalmitoyl}\text{-sn-}\text{glycerol-3-phosphoric}$ acid.The phase transition temperatures ($\text{T}{}_{\mathrm{<mtext>t</mtext>}}$) of aqueous phosphatidic acid dispersions at different degrees of dissociation were measured using fluorescence spectroscopy and 90° light scattering. The $\text{T}{}_{\mathrm{<mtext>t</mtext>}}$ values are comparable to the melting points of the solid phosphatidic acids in the fully protonated states, but large differences exist for the charged states.The $\text{T}{}_{\mathrm{<mtext>t</mtext>}}$ vs. pH diagrams of the four phosphatidic acids are quite similar and of a characteristic shape. Increasing ionisation results in a maximum value for the transition temperatures at pH 3.5 ($\text{p}\text{K}{}_1$). The regions between the first and the second $\text{p}\text{K}$ of the phosphatidic acids are characterised by only small variations in the transition temperatures (extended plateau) in spite of the large changes occurring in the surface charge of the membranes. The slope of the plateau is very shallow with increasing ionisation. A further decrease in the H⁺ concentration results in an abrupt change of the transition temperature. The slope of the $\text{T}{}_{\mathrm{<mtext>t</mtext>}}$ vs. pH diagram beyond $\text{p}\text{K}{}_2$ becomes very steep. This is the     

The presence of multiple cytochrome b proteins in succinate-cytochrome c reductase.

Two cytochrome $\text{b}$ proteins were isolated from succinate-cytochrome $\text{c}$ reductase and the cytochrome $\text{b-c}{}_1$ complex. Their molecular weights were determined to be 37,000 and 17,000 daltons by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Spectral properties and amino acid composition of these two proteins are reported in the paper.     

A NMR study of the ionization of fatty acids,fatty amines and N-acylamino acids incorporated in phosphatidylcholine vesicles

The ionization of fatty acids, fatty amines and $\text{N-}\text{acylamino}$ acids incorporated in phosphatidylcholine single-walled vesicles has been measured. The guest molecules have been specifically enriched with ¹³C and titrated by using NMR spectroscopy. The apparent p$\text{K}{}_{\mathrm{a}}$ of fatty acids in phosphatidylcholine bilayers is 7.2–7.4 and those of fatty amines are approx. 9.5. These p$\text{K}{}_{\mathrm{a}}$ values depend on many different parameters related to the structure of the lipid/ solution interface, to the composition of the aqueous medium and to the localization of the ionizable groups. A special sensitivity to the ionic strength and to the surface charge has been found. A positive surface charge decreases the p$\text{K}{}_{\mathrm{a}}$ value whereas a negative one increases it, the total range of variation being 2.5–3 units. In a qualitative macroscopic interpretation, it is proposed that p$\text{K}{}_{\mathrm{a}}$ is essentially determined by the low polarity of the lipidic matrix.     

Malate dehydrogenase. Kinetic studies with meso-tartrate and 2-keto-3-hydroxysuccinate, comparison of the mitochondrial and supernatant pig heart enzymes.

Initial rate, product inhibition, and isotope rate kinetic studies of pig heart mitochondrial and supernatant malate dehydrogenases, acting upon the nonphysiological substrates, meso-tartrate and 2-keto-3-hydroxysuccinate, are reported. The measured spontaneous keto-enol equilibrium for 2-keto-3-hydroxysuccinate in 0.05 m Tris-acetate (pH 8.0) at 25 °C favors the enol form, dihydroxyfumarate, with an apparent equilibrium constant of 0.036. The enzyme-catalyzed reaction favors meso-tartrate with an apparent equilibrium constant of 1.25 × 10^?6, M^?1 at pH 8.0. The mechanism apparently remains ordered bi bi for both enzymes when these nonphysiological substrates are used, and the chemical-converting hydride transfer step becomes more rate limiting for both enzymes. This conclusion is supported by $\text{V}{}_{\mathrm{<mtext>H</mtext>}}\text{V}{}_{\mathrm{<mtext>D</mtext>}}$ and $\text{(}\text{V}{}_{\mathrm{<mtext>H</mtext>}}\text{K}{}_{\mathrm{<mtext>H</mtext>}}\text{)}\text{V}{}_{\mathrm{<mtext>D</mtext>}}\text{K}{}_{\mathrm{<mtext>D</mtext>}}$ values of 2.6 and 3.1, respectively, for the mitochondrial enzyme and 1.9 and 2.9, respectively, for the supernatant enzyme.     

Sodium dependence of maximum flux, JM, and Km of amino acid transport in Ehrlich ascites cells

The Michaelis-Menten parameters, $\text{J}{}_{\mathrm{<mtext>M</mtext>}}$ and $\text{K}{}_{\mathrm{m}}$ of the initial 1-min fluxes of uptake of l-phenylalanine and of α-aminoisobutyric acid were determined for extracellular concentrations of Na⁺ ranging from 0.5 to 110 mequiv/l for Ehrlich ascites tumor cells. The maximal initial flux, $\text{J}{}_{\mathrm{<mtext>M</mtext>}}$, decreased with decrease in extracellular Na⁺ for both α-aminoisobutyric acid and phenylalanine but the $\text{K}{}_{\mathrm{m}}$ for α-aminoisobutyric acid increased markedly as the Na⁺ concentration fell whereas the $\text{K}{}_{\mathrm{m}}$ for phenylalanine decreased. Cycloleucine behaved like phenylalanine.The data provides strong evidence that the Na⁺-independent flux of phenylalanine is an exchange diffusion flux that can be varied by changing the intracellular level of amino acids such as phenylalanine. For phenylalanine, cyclolcucine, and methionine this exchange diffusion flux appears to be additive with the Na⁺-dependent initial flux. α-Aminoisobutyric acid also has an exchange diffusion that is Na⁺-independent but it has a high $\text{K}{}_{\mathrm{m}}$ and is not additive with the Na⁺-dependent flux.     

Oxidation of anionic nitroalkanes by flavoenzymes,and participation of superoxide anion in the catalysis

The reactivities of anionic nitroalkanes with 2-nitropropane dioxygenase of Hansenula mrakii, glucose oxidase of Aspergillus niger, and mammalian d-amino acid oxidase have been compared kinetically. 2-Nitropropane dioxygenase is 1200 and 4800 times more active with anionic 2-nitropropane than d-amino acid oxidase and glucose oxidase, respectively. The apparent K_m values for anionic 2-nitropropane are as follows: 2-nitropropane dioxygenase, 1.61 mm; glucose oxidase, 16.7 mm; and d-amino acid oxidase, 11.1 mm. Anionic 2-nitropropane undergoes an oxygenase reaction with 2-nitropropane dioxygenase and glucose oxidase, and an oxidase reaction with d-amino acid oxidase. In contrast, anionic nitroethane is oxidized through an oxygenase reaction by 2-nitropropane dioxygenase, and through an oxidase reaction by glucose oxidase. All nitroalkane oxidations by these three flavoenzymes are inhibited by Cu and Zn-superoxide dismutase of bovine blood, Mn-superoxide dismutases of bacilli, Fe-superoxide dismutase of Serratia marcescens, and other $\text{O}{}_2{}^{\mathrm{?}}$ scavengers such as cytochrome c and NADH, but are not affected by hydroxyl radical scavengers such as mannitol. None of the $\text{O}{}_2{}^{\mathrm{?}}$ scavengers tested affected the inherent substrate oxidation by glucose oxidase and d-amino acid oxidase. Furthermore, the generation of $\text{O}{}_2{}^{\mathrm{?}}$ in the oxidation of anionic 2-nitropropane by 2-nitropropane dioxygenase was revealed by ESR spectroscoy. The ESR spectrum of anionic 2-nitropropane plus 2-nitropropane dioxygenase shows signals at g₁ = 2.007 and g₁₁ = 2.051, which are characteristic of $\text{O}{}_2{}^{\mathrm{?}}$. The $\text{O}{}_2{}^{\mathrm{?}}$ generated is a catalytically essential intermediate in the oxidation of anionic nitroalkanes by the enzymes.     

Effects of L-methionine-DL-sulfoximine and beta-N-oxalyl-L-alpha, beta-diaminopropionic acid on nitrogenase biosynthesis and activity in Rhodopseudomonas capsulata.

Inhibitors of glutamine synthetase cause derepression of nitrogenase biosynthesis in the presence of $\text{NH}{}_4{}^{\mathrm{+}}$ in the photosynthetic bacterium Rhodopseudomonas capsulata. A new derepressor of nitrogenase biosynthesis, β-N-oxalyl-L-α,β-diaminopropionic acid (ODAP), is here compared with the widely used L-methionine-DL-sulfoximine (MSX). With both compounds, a quantitative correlation has been observed between inhibition of glutamine synthetase and derepression of nitrogenase biosynthesis. We also find that both MSX and ODAP inhibit nitrogenase activity in vivo in R. capsulata. The latter effect seems to be indirect and related to the previously reported reversible inhibition of nitrogenase activity in vivo by $\text{NH}{}_4{}^{\mathrm{+}}$. As a control it was observed that neither $\text{NH}{}_4{}^{\mathrm{+}}$ nor MSX nor ODAP inhibit nitrogenase activity in vivo in Clostridium pasteurianum.     

Enzymic and physicochemical characterization of ribulose 1,5-bisphosphate carboxylase/oxygenase from diploid and tetraploid cultivars of perennial ryegrass

Homogeneous preparations of ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) were isolated from several diploid and tetraploid cultivars of perennial ryegrass (Lolium perenne L.) by three different purification protocols. The apparent K_m values for substrate CO₂ were essentially identical for the fully $\text{CO}{}_2\text{Mg}{}^{\mathrm{2+}}$-activated diploid and tetraploid enzymes, as were the kinetics for deactivation and activation of the $\text{CO}{}_2\text{Mg}{}^{\mathrm{2+}}$ activated and -depleted carboxylases, respectively. Similarly, virtually indistinguishable electrophoretic properties were observed for both the native and dissociated diploid and tetraploid ryegrass proteins, including native and subunit molecular weights and the isoelectric points of the native proteins and the large and small subunit component polypeptides. The quantity of carboxylase protein or total soluble leaf protein did not differ significantly between the diploid and tetraploid cultivars. Contrary to a previous report (M. K. Garrett, 1978, Nature (London)274, 913–915), these results indicate that increased ploidy level (i.e., nuclear gene dosage) has had essentially no effect on the quantity or enzymic and physicochemical properties of ribulosebisphosphate carboxylase/ oxygenase in perennial ryegrass.     

The addition of bisulfite to 5-fluorouracil. Evidence for a change in rate determining step

The kinetics of bisulfite addition to 5-fluorouracil were studied as a function of increasing concentrations of potential general acids. Values of $\text{k}{}_{\mathrm{obsd}}\text{[}\text{SO}{}_3{}^{\mathrm{=}}\text{]}$ measured at 25°C and ionic strength 1.0 M increased linearly and then became invariant with increasing concentrations of either HSO₃^? or (OHCH₂CH₂)₂N⁺C(CH₂OH)₃ HCl (BisTris⁺HCl). A small kinetic hydrogen-deuterium isotope effect ($\text{k}{}_{\mathrm{HS}}\text{k}{}_{\mathrm{DS}}\text{= 1.10}$) was observed for the general acid catalysed portion of the addition reaction. The kinetics of bisulfite elimination from 5-fluoro-5,6-dihydrouracil-6-sulfonate were studied in ethanolamine buffers. As previously observed with 1,3-dimethyl-5,6-dihydrouracil-6-sulfonate, this reaction is subject to general base catalysis and exhibits a large kinetic hydrogen-deuterium isotope effect (). The kinetic results for the addition reaction are consistent with a multistep reaction pathway involving the initial formation of an oxyanion sulfite addition intermediate (II) which subsequently adds a proton and undergoes tautomerization to yield the final 5-fluoro-5,6-dihydrouracil-6-sulfonate product. Thus the elimination of bisulfite from 5-fluoro-5,6-dihydrouracil-6-sulfonate probably proceeds by an ElcB mechanism which involves, at relatively low concentrations of general base, rate determining general base catalyzed proton abstraction from carbon 5 to yield intermediate II followed by the rapid elimination of sulfite to yield 5-fluorouracil. These results may be related to both the enzymatically catalyzed dehalogenation of bromoand iodouracil and the methylation of deoxyuridylate by thymidylate synthetase.     

Superoxide anion as a cofactor of dopamine-beta-hydrxylase.

Superoxide anion can serve a reducing agent for tyramine hydroxylation by dopamine-β-hydroxylase. Stable $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ solutions were obtained by dissolving KO₂ in dry dimethylsulfoxide and infused into buffered solutions of tyramine and dopamine-β-hydroxylase at constant rate. The reaction requires molecular oxygen, but differs from the ascorbate dependent hydroxylation in its alkaline pH optimum value (pH 7.5) and its low rate (9 nmol octopamine formed/min/mg of protein). In absence of tyramine $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ does not produce a stable reduced form of the enzyme.     

The diffusion-concentration product of oxygen in lipid bilayers using the spin-label T1 method

A method is described to measure the oxygen diffusion-concentration product, $\text{D}{}_{\mathrm{<mtext>o</mtext>}}\text{[}\text{O}{}_2\text{]}$, at any locus that can be probed or labeled using nitroxide radicals. The method is based on the dependence of the spin-lattice relaxation time $\text{T}{}_1$ of the spin label on the bimolecular collision rate with oxygen. Strong Heisenberg exchange between spin label and oxygen contributes directly to $\text{T}{}_1$ of the spin label, while dipolar interactions are negligible. Both time-domain and continuous wave saturation methods for studying $\text{T}{}_1$ are considered. The method has been applied to phospholipid liposomes using fatty acid spin labels. A discontinuity in $\text{D}{}_{\mathrm{<mtext>o</mtext>}}\text{[}\text{O}{}_2\text{]}$ at the main phase transition was observed.     

Structural studies of a french bean storage protein: phaseolin

Molecular weights and sedimentation coefficients have been measured for different oligomeric forms of phaseolin, the major storage protein in seeds of Phaseolus vulgaris L. The results indicate that phaseolin is a trimer ($\text{M}{}_{\mathrm{r}}\text{= 150000}$) at neutral pH which aggregates further to a dodecamer form ($\text{M}{}_{\mathrm{r}}\text{= 596000}$) at pH 4.5. The subunit size is in good agreement with the recently determined sequence molecular weight, if allowance is made for bound oligosaccharide and phytic acid moieties. The trimeric nature at neutral pH has been confirmed by chemical crosslinking studies using dimethylsuberimidate and dithiobis(succinimidylpropionate). Analyses of optical rotatory dispersion and circular dichroism data have been used to examine the corformation of phaseolin. In common with other seed globulins, a low proportion of α-helix ($\text{～ 10\%}$) coupled with a high level of β-sheet ($\text{～50\%}$) is predicted. These data are compared with a structural analysis based on the amino acid sequence of a phaseolin subunit polypeptide. The predicted level of α-helix is increased ($\text{～20\%}$) when phaseolin is heated in sodium dodecyl sulphate, but not when the detergent is added at room temperature.     

Chemical and spectroscopic conformation of an exogenous ligand bridge in half met hemocyanin.

Half $\text{met-N}{}_3{}^{\mathrm{?}}$ hemocyanin is shown to undergo a unique change at the Cu(II)?Cu(I) active site with temperature, exhibiting class II mixed valent properties at low temperature (The appearance of an intense near IR intervalence-transfer transition and a delocalized EPR spectrum). This requires a Cu(II)NNNCu(I) bridging geometry. The effects of CO coordination to half $\text{met-N}{}_3{}^{\mathrm{?}}$, combined with the presence of a low energy $\text{N}{}_3{}^{\mathrm{?}}\text{→ Cu(II)}$ charge transfer transition, demonstrate that azide is also bridging at room temperature. Finally, half $\text{met-N}{}_3{}^{\mathrm{?}}$ is found to be capable of coordination of a second $\text{N}{}_3{}^{\mathrm{?}}$ at the copper(II) site.     

Effect of para-chloromercuribenzenesulfonic acid and temperature on cell water osmotic permeability of proximal straight tubules

The apparent Arrhenius energy of activation ($\text{E}{}_{\mathrm{<mtext>a</mtext>}}$) of the water osmotic permeability ($\text{P}{}_{\mathrm{<mtext>os</mtext>}}{}^{\mathrm{<mtext>c</mtext>}}$) of the basolateral plasma cell membrane of isolated rabbit proximal straight tubules has been measured under control conditions and after addition of 2.5 mM of the sulfhydryl reagent, para-chloromercuribenzenesulfonic acid (pCMBS), of mersalyl and of dithiothreitol. $\text{E}{}_{\mathrm{<mtext>a</mtext>}}$ (kcal/mol) was $\text{3.2 ± 1.4}$ (controls) and $\text{9.2 ± 2.2}$ (pCMBS), while $\text{P}{}_{\mathrm{<mtext>os</mtext>}}{}^{\mathrm{<mtext>c</mtext>}}$ decreased with pCMBS to $\text{0.26 ± 0.17}$ of its control value. Mersalyl also decreased $\text{P}{}_{\mathrm{<mtext>os</mtext>}}{}^{\mathrm{<mtext>c</mtext>}}$ both in vitro and in vivo (using therapeutical doses). These actions of pCMBS and mersalyl were quickly reverted with 5 mM dithiothreitol and prevented by 0.1 M thiourea. $\text{E}{}_{\mathrm{<mtext>a</mtext>}}$ for free viscous flow is 4.2 and greater than 10 for non-pore-containing lipid membranes. By analogy with these membranes and with red blood cells, where similar effects of pCMBS on $\text{P}{}_{\mathrm{<mtext>os</mtext>}}$ are observed, it is concluded that cell membranes of the proximal tubule are pierced by aqueous pores which are reversibly shut by pCMBS. Part of the action of mercurial diuretics can be explained by their action on $\text{P}{}_{\mathrm{<mtext>os</mtext>}}{}^{\mathrm{<mtext>c</mtext>}}$.